The major scientific focus of my laboratory is to determine the Ras-initiated signaling pathways and their relevant transcriptional targets that contribute to human epithelial cell transformation and metastasis. Ras is mutated in approximately one quarter of all human cancers with the highest incidence in pancreatic, lung, colon, and thyroid tumors. In addition, there is considerable experimental evidence that persistent upstream signaling in other epithelial cancers may activate Ras. Because multiple autocrine or paracrine growth factor pathways contribute to the transformation of epithelial cells and their colonization of distant tissues during the development of metastasis, Ras signaling pathways are expected to provide broadly applicable diagnostic markers and therapeutic targets. Multiple downstream effectors mediate Ras signaling, and there is a growing appreciation that the signaling outcomes of Ras activation demonstrate species and cell context differences. My laboratory has developed two model systems to address the mechanisms of Ras action. First, we have shown that ectopic Ras activation leads to the new expression of a bone metastatic phenotype in the DU145 xenograft model of human prostate cancer, and furthermore, that bone tropism can be mediated by a specific Ras effector pathway. There are very few models of metastatic prostate cancer, and our DU145 model is unique in that the initiating genetic event, i.e. Ras activation, can be manipulated. Second, we have developed a model of Ras-mediated transformation of immortalized breast epithelia that addresses the multiple and tissue-specific mechanisms of Ras signaling. The consequences of Ras activation are complex, resulting from the engagement of multiple downsteam effectors. The best-characterized effectors are Raf, PI3-kinase, and RalGEF, which have been shown to account for many of the biological consequences of Ras activation, although clearly, additional Ras-interacting proteins will play a role. As one approach, we have used activated Ras effector mutants, Ras12V35S (RasS35), Ras12V37G (RasG37), and Ras12V40C (RasC40) that relatively uniquely activate Raf, RalGEF, and PI3-kinase, respectively, to broadly dissect the relationship of signaling pathways and biological function mediated by Ras. My laboratory was one of the first to identify the RasG37 and RalGEF pathways as playing a role in metastasis using a system of experimental lung metastasis and 3T3 cells. We have extended these results to show in the DU145 model that Ras effector mutants influence the tissue tropism of metastasis. RasC40 expressing cells show predominantly a tropism for brain, while RasG37 expressing cells demonstrate a high efficiency of bone metastases, the most common site of metastasis for advanced prostate cancer in patients. RasS35 transformed cells metastasize to soft tissue, brain, and bone. We are using RasG37 transformed DU145 cells to analyze the interaction of prostate cancer cells with the bone microenvironment as it has the advantage of being selective with respect to both signaling pathway and tissue specificity. We have used gene profiling to investigate potentially important autocrine and paracrine effects of RasG37 activation. RasG37 appears to alter responses to TGFb, a major component of the bone environment and a common autocrine factor in prostate cancer, by increasing the expression of factors that favor tumor cell growth in the bone and by decreasing growth inhibitory responses. The DU145(Ras) system provides an experimental system for the identification of genes mediating metastasis to different organs. It also provides an efficient and predictable model system of tissue-specific metastases for preclinical testing of therapeutic agents.Recent data suggest that Ras may transform murine and human cells by distinct mechanisms and that the RasG37/RalGEF pathway is the most prominent effector pathway mediating human cell transformation.